Gram-Positive Bacteria Cell Wall Peptidoglycan Polymers Activate Human Dendritic Cells to Produce IL-23 and IL-1ß and Promote TH17 Cell Differentiation.

Gram-positive bacterial infections are a major cause of organ failure and mortality in sepsis. Cell wall peptidoglycan (PGN) is shed during bacterial replication, and Bacillus anthracis PGN promotes a sepsis-like pathology in baboons. Herein, we determined the ability of polymeric Bacillus anthracis PGN free from TLR ligands to shape human dendritic cell (DC) responses that are important for the initiation of T cell immunity. Monocyte-derived DCs from healthy donors were incubated with PGN polymers isolated from Bacillus anthracis and Staphylococcus aureus. PGN activated the human DCs, as judged by the increased expression of surface HLA-DR, CD83, the T cell costimulatory molecules CD40 and CD86, and the chemokine receptor CCR7. PGN elicited the DC production of IL-23, IL-6, and IL-1ß but not IL-12p70. The PGN-stimulated DCs induced the differentiation of naïve allogeneic CD4+ T cells into T helper (TH) cells producing IL-17 and IL-21. Notably, the DCs from a subset of donors did not produce significant levels of IL-23 and IL-1ß upon PGN stimulation, suggesting that common polymorphisms in immune response genes regulate the PGN response. In sum, purified PGN is a highly stimulatory cell wall component that activates human DCs to secrete proinflammatory cytokines and promote the differentiation of TH17 cells that are important for neutrophil recruitment in extracellular bacterial infections.

Toxin inhibition of antimicrobial factors induced by Bacillus anthracis peptidoglycan in human blood.

Here, we describe the capacity of Bacillus anthracis peptidoglycan (BaPGN) to trigger an antimicrobial response in human white blood cells (WBCs). Analysis of freshly isolated human blood cells found that monocytes and neutrophils, but not B and T cells, were highly responsive to BaPGN and produced a variety of cytokines and chemokines. This BaPGN-induced response was suppressed by anthrax lethal toxin (LT) and edema toxin (ET), with the most pronounced effect on human monocytes, and this corresponded with the higher levels of anthrax toxin receptor 1 (ANTXR1) in these cells than in neutrophils. The supernatant from BaPGN-treated cells altered the growth of B. anthracis Sterne, and this effect was blocked by LT, but not by ET. An FtsX mutant of B. anthracis known to be resistant to the antimicrobial effects of interferon-inducible Glu-Leu-Arg (ELR)-negative CXC chemokines was not affected by the BaPGN-induced antimicrobial effects. Collectively, these findings describe a system in which BaPGN triggers expression of antimicrobial factors in human WBCs and reveal a distinctive role, not shared with ET, in LT's capacity to suppress this response.

Bacillus anthracis peptidoglycan activates human platelets through FcγRII and complement.

Platelet activation frequently accompanies sepsis and contributes to the sepsis-associated vascular leakage and coagulation dysfunction. Our previous work has implicated peptidoglycan (PGN) as an agent causing systemic inflammation in gram-positive sepsis. We used flow cytometry and fluorescent microscopy to define the effects of PGN on the activation of human platelets. PGN induced platelet aggregation, expression of the activated form of integrin αIIbß3, and exposure of phosphatidylserine (PS). These changes were dependent on immunoglobulin G and were attenuated by the Fcγ receptor IIa-blocking antibody IV.3, suggesting they are mediated by PGN-anti-PGN immune complexes signaling through Fcγ receptor IIa. PS exposure was not blocked by IV.3 but was sensitive to inhibitors of complement activation. PGN was a potent activator of the complement cascade in human plasma and caused deposition of C5b-9 on the platelet surface. Platelets with exposed PS had greatly accelerated prothrombinase activity. We conclude that PGN derived from gram-positive bacteria is a potent platelet agonist when complexed with anti-PGN antibody and could contribute to the coagulation dysfunction accompanying gram-positive infections.

Anti-peptidoglycan antibodies and Fcγ receptors are the key mediators of inflammation in Gram-positive sepsis.

Gram-positive bacteria are an important public health problem, but it is unclear how they cause systemic inflammation in sepsis. Our previous work showed that peptidoglycan (PGN) induced proinflammatory cytokines in human cells by binding to an unknown extracellular receptor, followed by phagocytosis leading to the generation of NOD ligands. In this study, we used flow cytometry to identify host factors that supported PGN binding to immune cells. PGN binding required plasma, and plasma from all tested healthy donors contained IgG recognizing PGN. Plasma depleted of IgG or of anti-PGN Abs did not support PGN binding or PGN-triggered cytokine production. Adding back intact but not F(ab')2 IgG restored binding and cytokine production. Transfection of HEK293 cells with FcγRIIA enabled PGN binding and phagocytosis. These data establish a key role for anti-PGN IgG and FcγRs in supporting inflammation to a major structural element of Gram-positive bacteria and suggest that anti-PGN IgG contributes to human pathology in Gram-positive sepsis.

Retromer guides STxB and CD8-M6PR from early to recycling endosomes, EHD1 guides STxB from recycling endosome to Golgi.

Retrograde trafficking transports proteins, lipids and toxins from the plasma membrane to the Golgi and endoplasmic reticulum (ER). To reach the Golgi, these cargos must transit the endosomal system, consisting of early endosomes (EE), recycling endosomes, late endosomes and lysosomes. All cargos pass through EE, but may take different routes to the Golgi. Retromer-dependent cargos bypass the late endosomes to reach the Golgi. We compared how two very different retromer-dependent cargos negotiate the endosomal sorting system. Shiga toxin B, bound to the external layer of the plasma membrane, and chimeric CD8-mannose-6-phosphate receptor (CI-M6PR), which is anchored via a transmembrane domain. Both appear to pass through the recycling endosome. Ablation of the recycling endosome diverted both of these cargos to an aberrant compartment and prevented them from reaching the Golgi. Once in the recycling endosome, Shiga toxin required EHD1 to traffic to the TGN, while the CI-M6PR was not significantly dependent on EHD1. Knockdown of retromer components left cargo in the EE, suggesting that it is required for retrograde exit from this compartment. This work establishes the recycling endosome as a required step in retrograde traffic of at least these two retromer-dependent cargos. Along this pathway, retromer is associated with EE to recycling endosome traffic, while EHD1 is associated with recycling endosome to TGN traffic of STxB.

Ca2+ chemotaxis in Dictyostelium discoideum.

Using a newly developed microfluidic chamber, we have demonstrated in vitro that Ca(2+) functions as a chemoattractant of aggregation-competent Dictyostelium discoideum amoebae, that parallel spatial gradients of cAMP and Ca(2+) are more effective than either alone, and that cAMP functions as a stronger chemoattractant than Ca(2+). Effective Ca(2+) gradients are extremely steep compared with effective cAMP gradients. This presents a paradox because there is no indication to date that steep Ca(2+) gradients are generated in aggregation territories. However, given that Ca(2+) chemotaxis is co-acquired with cAMP chemotaxis during development, we speculate on the role that Ca(2+) chemotaxis might have and the possibility that steep, transient Ca(2+) gradients are generated during natural aggregation in the interstitial regions between cells.

MAP kinases have different functions in Dictyostelium G protein-mediated signaling.

Extracellular signal regulated kinases (ERKs) are a class of MAP kinases that function in many signaling pathways in eukaryotic cells and in some cases, a single stimulus can activate more than one ERK suggesting functional redundancy or divergence from a common pathway. Dictyostelium discoideum encodes only two MAP kinases, ERK1 and ERK2, that both function during the developmental life cycle. To determine if ERK1 and ERK2 have overlapping functions, chemotactic and developmental phenotypes of erk1(-) and erk2(-) mutants were assessed with respect to G protein-mediated signal transduction pathways. ERK1 was specifically required for Galpha5-mediated tip morphogenesis and inhibition of folate chemotaxis but not for cAMP-stimulated chemotaxis or cGMP accumulation. ERK2 was the primary MAPK phosphorylated in response to folate or cAMP stimulation. Cell growth was not altered in erk1(-), erk2(-) or erk1(-)erk2(-) mutants but each mutant displayed a different pattern of cell sorting in chimeric aggregates. The distribution of GFP-ERK1 or GFP-ERK2 fusion proteins in the cytoplasm and nucleus was not grossly altered in cells stimulated with cAMP or folate. These results suggest ERK1 and ERK2 have different roles in G protein-mediated signaling during growth and development.

G{alpha}5 subunit-mediated signalling requires a D-motif and the MAPK ERK1 in Dictyostelium.

The Dictyostelium Galpha5 subunit has been shown to reduce cell viability, inhibit folate chemotaxis and accelerate tip morphogenesis and gene expression during multicellular development. Alteration of the D-motif (mitogen-activated protein kinase docking site) at the amino terminus of the Galpha 5 subunit or the loss of extracellular signal-regulated kinase (ERK)1 diminished the lethality associated with the overexpression or constitutive activation of the Galpha5 subunit. The amino-terminal D-motif of the Galpha5 subunit was also found to be necessary for the reduced cell size, small aggregate formation and precocious developmental gene expression associated with Galpha5 subunit overexpression. This D-motif also contributed to the aggregation delay in cells expressing a constitutively active Galpha5 subunit, but the D-motif was not necessary for the inhibition of folate chemotaxis. These results suggest that the amino-terminal D-motif is required for some but not all phenotypes associated with elevated Galpha5 subunit functions during growth and development and that ERK1 can function in Galpha5 subunit-mediated signal transduction.

A cAMP receptor-like G protein-coupled receptor with roles in growth regulation and development.

Dictyostelium discoideum uses G protein-mediated signal transduction for many vegetative and developmental functions, suggesting the existence of G protein-coupled receptors (GPCRs) other than the four known cyclic adenosine monophosphate (cAMP) receptors (cAR1-4). Sequences of the cAMP receptors were used to identify Dictyostelium genes encoding cAMP receptor-like proteins, CrlA-C. Limited sequence identity between these putative GPCRs and the cAMP receptors suggests the Crl receptors are unlikely to be receptors for cAMP. The crl genes are expressed at various times during growth and the developmental life cycle. Disruption of individual crl genes did not impair chemotactic responses to folic acid or cAMP or alter cAMP-dependent aggregation. However, crlA(-) mutants grew to a higher cell density than did wild-type cells and high-copy-number crlA expression vectors were detrimental to cell viability, suggesting that CrlA is a negative regulator of cell growth. In addition, crlA(-) mutants produce large aggregates with delayed anterior tip formation indicating a role for the CrlA receptor in the development of the anterior prestalk cell region. The scarcity of GFP-expressing crlA(-) mutants in the anterior prestalk cell region of chimeric organisms supports a cell-autonomous role for the CrlA receptor in prestalk cell differentiation.